The present invention relates to analytical chemistry, and in particular relates to sample preparation for molecular analysis.
In order to carry out molecular analysis (the task of identifying one or more compounds in a sample) of any product, a sample of the product must be in such a form that it can be easily analyzed by chromatography, spectroscopy, mass spectroscopy and/or nuclear magnetic resonance instrumentation
Because these analytical instruments require substantially pure isolated analytes, some intermediate steps, generally referred to as “sample preparation”, must be carried out to isolate the compounds of interest from the sample matrix in which they might be found and prepare them for analysis by instrumentation.
The task of identifying one or more compounds in a sample—presents an enormously larger set of possibilities and challenges related to sample preparation. The number of “naturally occurring” compounds (those produced by plants or animals) is immeasurably large, and the capabilities of modern organic and inorganic synthesis have generated—figuratively or literally—a similar number of synthetic compounds.
There is tremendous interest in identification or quantitative measurement for compounds of interest as it relates to industrial processes, and for environmental testing for contaminants in waste water, soil, and air.
Even a small group of recognizable representative samples would include pesticides in food, other synthetic chemicals in food (antibiotics, hormones, steroids), synthetic compositions (benzene, toluene, refined hydrocarbons) in soil, and undesired compositions in everyday items (e.g., Bisphenol-A (“BPA”) in polycarbonate bottles and other plastic food packaging.
In general extraction has been a main form of sample preparation; i.e., drawing one or more compounds of interest from a sample by mixing the sample with a solvent into which the desired compound(s) will be extracted from the sample so that it can be measured by an analytical technique.
For several generations (and continuing to date), sample preparation in the form of extraction has been carried out by the well-understood Soxhlet method which was invented in the 19th century. In the Soxhlet technique, a single portion of solvent circulates repeatedly through a sample matrix until extraction is complete. To the extent the Soxhlet method has an advantage, it allows an extraction to continue on its own accord for as long as the boiling flask is heated and the condenser is cold.
This method of extraction can take hours to completely extract the compounds of interest. Other concerns of safety from flammable solvents, hazardous waste and breakable glassware are significant drawbacks to this method.
Another commonly known extraction method uses ultra-sonication; i.e., the irradiation of a liquid sample with ultrasonic (>20 kHz) waves resulting in agitation. Although ultra-sonication has an advantage of speeding up the extraction process the disadvantages are that it is a labor intensive, manual process and uses large amounts of solvents.
In more recent years analytical scale microwave-assisted extraction (MAE) has been utilized. MAE uses microwave energy to heat solvents in contact with a sample in order to partition analytes from the sample matrix into the solvent. The main advantage of MAE is the ability to rapidly heat the sample solvent mixture. When using closed pressurized-vessels the extraction can be performed at elevated temperatures that accelerate the extraction of the compounds of interest from the sample matrix. MAE accelerates the extraction process, but has its disadvantages as well. In the microwave heating process typically a polar solvent is needed to provide dipole rotation and ionic conduction through reversals of dipoles and displacement of charged ions present in the solute and the solvent, limiting non-polar solvent use. MAE uses expensive, high-pressure vessels that do not provide a means of filtering the extract, and they must be cooled before pressure can be released.
In the 1990's automated apparatuses for the extraction of analytes were developed. These apparatuses incorporated solvent extraction in pressurized cells under elevated temperatures and pressures and are referred to as “Pressurized Fluid Extraction” (“PFE”) or “Accelerated Solvent Extraction” (“ASE”). PFE has shown to be similar to Soxhlet extraction, except that the solvents are at elevated temperatures where they exhibit high extraction properties. This procedure was first developed by Dionex (Richter D E et al., Anal Chem 1996, 68, 1033). One such PFE automated extraction system (Dionex ASE) is commercially available.
PFE was initially used for environmental contaminants (EPA Method 3545, herbicides, pesticides, hydrocarbons) in soil, sediments and animal tissues but has expanded to use in foods, pharmaceutical products and other biological samples.
PFE provides an efficient extraction, but still has not overcome the major bottlenecks associated with the many steps necessary to prepare a sample for analysis. PFE utilizes multiple-component cells and many steps. The cells are tightly packed with the sample and other packing material to eliminate any void areas in the cell, enhance separation, and avoid channeling. Preparing a cell for analysis can typically take 15 minutes. The cells are pre-pressurized at pressures up to 1500 psi and heated up to 200° C. prior to adding the solvent. Extraction is based on chromatographic principles to force the hot solvent through the column. Cycle times can take up to 20 minutes and the requirements of high pressure lead to secondary disadvantages with respect to cost and maintenance.
Newer PSE or ASE techniques attempt to address some of these difficulties, but still require that the cells be tightly packed, adding to the complexity and overall time required for each extraction.
Sample preparation, although having developed over the years, nevertheless remains the major bottleneck in molecular analysis. Accordingly, although the Soxhlet, Ultrasonication, MAE and PFE techniques have their advantages, each remains relatively time-consuming. As a result, when multiple samples are required or desired to provide necessary or desired information, the time required to carry out any given extraction-based molecular preparation step reduces the number of samples that can be prepared in any given amount of time, thus reducing the amount of information available in any given time interval. To the extent that measurements are helpful or necessary in a continuous process, this represents a longer gap between samples or before an anomalous or troublesome result can be identified.
In summary, current sample preparation techniques are slow, complex, inefficient, require a large number of separate steps, use excess solvent, are difficult to automate, and operate under high liquid pressure.
Accordingly, extraction-based sample preparation continues to be recognized as a major bottleneck in analytical techniques.